Major efforts in functional genomics and proteomics are creating an unprecedented demand for monoclonal antibodies to be used for protein function studies. Monoclonal antibodies can be used in every stage of research involving protein target discovery and characterization including purification, quantification, and organ and cellular localization. Recent advances in proteomics are creating a need for large numbers of antibodies for use in high throughput studies and on protein chips. Monoclonal antibodies have been used for decades as key reagents in clinical diagnostics and they are emerging as an important new class of therapeutics agents.
Hybridoma technology is the most commonly used method for accessing monoclonal antibodies. Monoclonal antibodies are secreted from hybridoma cells, created by fusing normal antibody producing splenic B-cells with immortal myeloma cells or other immortal cells. Hybridoma production has changed little since its inception 26 years ago (Kohler and Milstein, 1975).
A typical protocol for hybridoma generation involves: (i) immunizing an animal (e.g., mouse, rat or rabbit) with a purified protein antigen; (ii) harvesting antibody producing B-cells, typically from the spleen; (iii) fusing B-cells with a non-secretory myeloma cell line deficient for the enzyme hypoxanthine guanine phosphoribosyl transferase (e.g., x63-Ag 8.653 from a BALB/c mouse strain); (iv) growing hybridoma cells in a selection medium containing hypoxanthine, aminopterin and thymidine (HAT) and (v) screening for cells that produce the desired antibody and (vi) limit dilution cloning to obtain a homogenous cell line that secretes the antibody (Antczak, 1982).
Conventional hybridoma technology does not allow researchers to access large numbers of antibodies to different antigens or large numbers of antibodies to a single target antigen in an efficient manner. Hybridoma cell cloning by limit dilution is perhaps the most problematic, time consuming and labor intensive step in generating monoclonal antibodies (O'Reilly et.al., 1998). In this step, cells are repeatedly diluted out to low cell numbers and their supernatants assayed for secreted monoclonal antibody. Screening must be artfully performed to ensure that desired hybridomas are not lost. In the case of rapidly growing hybridomas, cells may die in the microtiter wells from exhaustion of nutrients if they are not moved to larger vessels or fresh medium quickly. Also, in typical wells containing several hybridomas, undesirable hybridomas may continuously overgrow desired hybridomas. This can cause the limit dilution step to be extended weeks or months and may even result in loss of important hybridomas. If the hybridomas have not grown to a reasonable size by the time of assay, they may not have produced sufficient antibody for detection. Therefore, a time for screening supernatants must be chosen carefully. The available “window” for initial screening is not large and usually extends over two to three days (Antczak, 1982). Once started, the limit dilution isolation of pure cell lines typically goes on for 3–4 weeks for any one hybridoma.
There is a need for more rapid methods of isolating desired hybridoma cells. At least two laboratories attempted to sort normal hybridomas based on traces of surface presentation of antibody (Parks et al., 1979; Meilhoc et al., 1989). They showed that a subset of hybridoma cells in any population presented a small but measurable number (˜20) of surface antibody molecules. This was enough to sort these cells when labeled with antigen, if the antigen was coupled to highly fluorescent microspheres to increase the fluorescence signal. Even with these highly fluorescent spheres the signal was only a few-fold above the background.
There is a need for a significant increase in the presentation of surface antibody on hybridoma cells as well as a need for a significant increase in the percentage of hybridoma cells presenting surface antibody in any population to enable rapid screening. The present invention provides both by providing DISH (Direct Screening of Hybridoma Cells). The DISH technology of the present invention provides a simple, rapid and reliable selection of hybridoma cells that may be accomplished in a matter of hours instead of weeks. DISH provides several significant improvements over conventional hybridoma technology that allow researchers to access much larger repertoires of antibodies in an efficient manner. Using current hybridoma protocols approximately 40,000 fusions are typically prepared. A main reason more fusions are not made is the difficulty encountered in isolating desired clones using limit dilution. DISH enables very rapid, high throughput cell selection using fluorescence activated cell sorting (FACS) and other modalities. Since FACS technology permits millions of cells to be sorted in a matter of hours, the number of hybridoma fusions one can screen using DISH technology is orders of magnitude larger than by limit dilution. Significantly, FACS sorting allows for single cell deposition of desired hybridomas into discrete wells. Hence, the problem of desirable, but slow growing cells being lost is eliminated using DISH. Thus, DISH replaces current antibody screening and limit dilution procedures with a rapid, high throughput, selection process. The present invention can also be utilized to provide populations of plasma cells that surface present adequate immunoglobulin to enable high throughput fluorescence activated cell sorting technology to be used to determine whether single plasma cells produce immunoglobin that reacts with target antigens.